Recent accomplishments related to our immunotherapy clinical trials include the following: [] We recently reported the clinical results of a Phase I trial combining ipilimumab with a vaccine containing transgenes for prostate-specific antigen (PSA) and for a triad of costimulatory molecules (PROSTVAC) in patients with metastatic castration-resistant prostate cancer. Thirty patients were treated with escalating ipilimumab and a fixed dose of vaccine. Of 24 chemotherapy-naive patients, 58% had a PSA decline. Combination therapy did not exacerbate the immune-related adverse events associated with ipilimumab. We have now presented updated survival data and an evaluation of 36 immune cell subsets pre- and post-therapy. Peripheral blood mononuclear cells were collected before therapy, at 13 days and at 70 days post-initiation of therapy, and phenotyped by flow cytometry for the subsets of T cells, regulatory T cells, natural killer cells, and myeloid-derived suppressor cells. Associations between overall survival (OS) and immune cell subsets prior to treatment, and the change in a given immune cell subset 70 days post-initiation of therapy, were evaluated. There were clear trends toward associations for longer OS and several immune cell subsets before immunotherapy. These results should be considered as hypothesis generating and should be further evaluated in larger immunotherapy trials. [] Preclinical studies have demonstrated that the combination of systemic subcutaneous (s.c.) vaccination with intratumoral (i.t.) vaccination was superior in the induction of antitumor activity vs. vaccination with either route alone. A subsequent phase I study employing i.t.-s.c. vaccination was carried out in men with locally recurrent or progressive prostate cancer. rF-PSA-TRICOM (PROSTVAC) vaccine was administered intraprostatically and rV-PSA-TRICOM followed by rF-PSA-TRICOM vaccine was administered systemically. 19/21 patients had stable or improved PSA values and tumor-infiltrating lymphocytes (TILs) increased in post- vs. pre-treatment tumor biopsies, analyzed employing conventional immunohistochemistry (IHC). In these recent studies, 31 phenotypes of peripheral blood mononuclear cells (PBMCs) were analyzed pre-vaccination and post-vaccination as well as the functions of PBMC regulatory T cells (Tregs) and natural killer cells. A trend was observed in decreases in serum PSA with the reduction of circulating Tregs post-vaccination. Digital IHC was employed pre-vaccination and post-vaccination to measure CD4 and CD8 TILs, as well as Treg TILs by conventional IHC. Patients with lower levels of CD4 TILs pre-vaccination showed the greatest increases in CD4 TILs post-vaccine, while Treg TILs decreased post-vaccine. There was also a strong correlation between decreases in serum PSA and increases in CD8 TILs post-vaccine. These studies provide additional rationale for the use of i.t.-s.c. vaccinations and demonstrate a non-coordinate expression of specific immune subsets in PBMCs vs. tumor. [] PROSTVAC is a novel vector-based vaccine designed to generate a robust immune response against PSA-expressing tumor cells. The purpose of this study was to present an overview evaluation of immune responses to the PSA-TRICOM vaccine platform, currently in phase III testing. Of 104 patients tested for T-cell responses, 57% (59/104) demonstrated a = 2-fold increase in PSA-specific T cells after vaccine (median 5-fold increase) compared with pre-vaccine, and 68% (19/28) of patients tested mounted post-vaccine immune responses to tumor-associated antigens not present in the vaccine (antigen spreading). [] The goal of this study was to determine if the combination of docetaxel and PANVAC vaccine could provide evidence of improved clinical outcomes in patients with metastatic breast cancer vs. docetaxel alone. This open-label randomized Phase II, dual-center trial was designed to enroll 48 patients with metastatic breast cancer to receive docetaxel with PANVAC (arm A) or 38 docetaxel alone (arm B). This pilot study was powered to detect a trend of approximately this magnitude toward improvement in PFS. The results suggest the combination of PANVAC with docetaxel in metastatic breast cancer may provide a clinical benefit. This study was hypothesis generating and provides both rationale and statistical assumptions for a larger definitive randomized study. [] The nuclear transcription factor brachyury has previously been shown to be a strong mediator of the epithelial-to-mesenchymal transition (EMT) in human carcinoma cells and a strong negative prognostic factor in several tumor types. Brachyury is overexpressed in a range of human carcinoma as well as in chordoma, a rare tumor for which there is no standard systemic therapy. Preclinical studies have shown a recombinant Saccharomyces cerevisiae (yeast) vaccine encoding brachyury (GI-6301) can activate human T cells in vitro. A Phase I dose escalation trial enrolled 34 patients at 4 dose levels. Expansion cohorts were enrolled for analysis of immune response and clinical activity. We observed brachyury-specific T-cell immune responses in the majority of evaluable patients despite most having been heavily pretreated. No evidence of autoimmunity or other serious adverse events were observed. Two chordoma patients showed evidence of disease control (one mixed response and one partial response). A patient with colorectal carcinoma, who enrolled on study with a large progressing pelvic mass and rising carcinoembryonic antigen (CEA), remains on study for greater than 1 year with stable disease, evidence of decreased tumor density and decreased serum CEA. This study is the first-in-human to demonstrate the safety and immunogenicity of this therapeutic cancer vaccine and provides rationale for exploration in Phase II studies. A randomized Phase II chordoma study is enrolling.